Pacchia A L, Adelson M E, Kaul M, Ron Y, Dougherty J P
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
Virology. 2001 Mar 30;282(1):77-86. doi: 10.1006/viro.2000.0787.
Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.
基于1型人类免疫缺陷病毒（HIV-1）的慢病毒载体具有将外源基因传递至分裂细胞和非分裂细胞，并随后在这些靶细胞中建立稳定前病毒的能力，这能够使转移基因长期表达。在此，我们描述了一种稳定的包装细胞系，该细胞系缺乏HIV-1的tat、vif、vpr、vpu和nef基因。为了避免与HIV-1蛋白酶或VSV-G包膜蛋白的组成型表达相关的任何细胞毒性风险，通过采用蜕皮激素诱导系统严格控制包装和包膜构建体的转录。使用该细胞系，我们能够持续产生滴度在10（8）IU/ml范围内的浓缩假型载体病毒储备液，其能够在体外有效转导活跃分裂和生长停滞的细胞。这种用于慢病毒载体的新型包装细胞系有助于在无复制能力病毒的情况下生产高滴度病毒储备液，并为我们未来的基因转移研究提供了一个重要工具。
